A general approach to cancer therapy and diagnosis involves directing antibodies or antibody fragments to disease tissues, whereby the antibody or antibody fragment can target a diagnostic agent or therapeutic agent to the disease site. One specific approach to this methodology which has been under investigation, involves the use of bsAbs having at least one arm that specifically binds a targeted diseased tissue and at least one other arm that specifically binds a low molecular weight hapten. In this methodology, a bsAb is administered and allowed to localize to a target and to clear normal tissue. Some time later, a radiolabeled low molecular weight hapten is given, which, being recognized by the second specificity of the bsAb, also localizes to the original target.
Although low MW haptens used in combination with bsAbs possess a large number of specific imaging and therapy uses, it is impractical to prepare individual bsAbs for each possible application. Further, the application of a bsAb/low MW hapten system has several other requirements. First, the arm of the bsAb that binds to the low MW hapten must bind with high affinity, because a low MW hapten is designed to clear the living system rapidly when not bound by bsAb. Second, the non-bsAb-bound low MW hapten actually needs to clear the living system rapidly to avoid non-target tissue uptake and retention. Third, the detection and/or therapy agent must remain associated with the low MW hapten throughout its application within the bsAb protocol.
Of interest with this approach are bsAbs that direct chelators and metal chelate complexes to cancers using Abs of appropriate dual specificity. The chelators and metal chelate complexes used are often radioactive, using radionuclides for radioimmuno-imaging. (See Goodwin et al., U.S. Pat. No. 4,863,713 (describing the use of cobalt-57); Barbet et al., U.S. Pat. No. 5,256,395 and U.S. Pat. No. 5,274,076; Goodwin et al., J. Nucl. Med., 33:1366-1372 (1992); and Kranenborg et al., Cancer Res (suppl.), 55:5864s-5867s (1995) and Cancer (suppl.) 80:2390-2397 (1997) (all describing the use of indium-111); and Boden et al., Bioconjugate Chem., 6:373-379, (1995); and Schuhmacher et al., Cancer Res., 55:115-123 (1995)(describing the use of gallium-68)). Because the Abs are raised against the chelators and metal chelate complexes, they have remarkable specificity for the complex against which they were originally raised. Indeed, the bsAbs of Boden et al. have specificity for single enantiomers of enantiomeric mixtures of chelators and metal-chelate complexes. This great specificity has proven to be a disadvantage in one respect, in that other nuclides (such as yttrium-90 and bismuth-213 useful for radioimmunotherapy (RAIT), and gadolinium useful for MRI), cannot be readily substituted into available reagents for alternative uses. As a result iodine-131, a non-metal, has been adopted for RAIT purposes by using an I-131-labeled indium-metal-chelate complex in the second targeting step. Another disadvantage to this methodology requires that antibodies be raised against every agent desired for diagnostic or therapeutic use.
As such, pretargeting methodologies have received considerable attention for cancer imaging and therapy. Unlike direct targeting systems where an effector molecule (e.g., a radionuclide or a drug linked to a small carrier) is directly linked to the targeting agent (e.g., a binding molecule such as a bsAb), in pretargeting systems, the effector molecule is given some time after the targeting agent. This allows time for the targeting agent to localize in tumor lesions and, more importantly, clear from the body. Because most targeting agents have been binding proteins such as antibodies, they tend to clear much more slowly from the body (usually days) than the smaller effector molecules (usually in minutes). As such, in direct targeting systems involving therapeutic radionuclides, the body, and in particular the highly vulnerable red marrow, may be exposed to the radiation all the while the targeting agent is slowly reaching its peak levels in the tumor and clearing from the body. However, in a pretargeting system, the radionuclide (i.e., an effector) is usually bound to a small “carrier” molecule, such as a chelate or peptide, which clears very quickly from the body, and thus exposure of normal tissues is minimized. In a pretargeting system, maximum tumor uptake of the radionuclide is also very rapid because the small carrier molecule efficiently transverses the tumor vasculature and binds to the primary targeting agent. The small size of a carrier molecule may also encourage a more uniform distribution in the tumor.
Pretargeting methods have used a number of different strategies, but often involve an avidin/streptavidin-biotin recognition system or bi-specific antibodies that co-recognize a tumor antigen and one or mole haptens on the carrier molecule, which includes an effector molecule. The avidin/streptavidin system is highly versatile and has been used in several configurations. In this system, antibodies coupled with streptavidin or biotin are used as the primary targeting agent. This is followed sometime later by administration of the effector molecule, which may be conjugated with biotin or with avidin/streptavidin, respectively. Another configuration relies on a 3-step approach: (1) first targeting a biotin-conjugated antibody; (2) followed by a bridging with streptavidin/avidin; and (3) then the biotin-conjugated effector is given. These systems can be easily converted for use with a variety of effector substances so long as the effector and the targeting agent can be coupled with biotin or streptavidin/avidin depending on the configuration used. With its versatility for use in many targeting situations and high binding affinity between avidin/streptavidin and biotin, this type of pretargeting has considerable advantages over other proposed systems. However, avidin and streptavidin are foreign proteins and therefore can be immunogenic, which may limit the number of times they can be administered in a clinical application. In this respect, bsAbs have the advantage of being able to be engineered as a relatively non-immunogenic humanized protein. Although the binding affinity of a bsAb (typically 10−9 to 10−10 M) cannot compete with the extremely high affinity of the streptavidin/avidin-biotin affinity (˜10−15 M), both pretargeting systems are dependent on the binding affinity of the primary targeting agent, and therefore the higher affinity of the streptavidin/avidin-biotin systems may not offer a substantial advantage over a bsAb pretargeting system. However, most bsAbs have only one arm available for binding the primary target, whereas the streptavidin/avidin-biotin pretargeting systems typically use a whole IgG with two arms for binding the target, which strengthens target binding. By using a divalent peptide, an affinity enhancement may be achieved, which can greatly improve the binding of the peptide to the target site compared to a monovalent peptide. Thus, both systems can provide excellent targeting ratios with reasonable retention.
Pretargeting with a bsAb also requires one arm of the antibody to recognize an effector molecule or a molecule that contains an effector molecule (e.g., a carrier with an effector together as a “targetable construct”). Most radionuclide targeting systems reported to date have relied on an antibody to a chelate-metal complex, such as antibodies directed against indium-loaded DTPA or antibodies to other chelates. Because the antibody is generally selective for a particular chelate-metal complex, new bsAbs typically need to be constructed for each selected chelate-metal complex. This can be avoided by using a carrier molecule that includes the effector molecule and a hapten, which is specifically recognized by the antibody. As such, the carrier, including the effector and hapten, functions as a targetable construct. The targetable construct is “modular” in nature, in that different effectors can be included in the construct without having to use a different antibody in the pretargeting system, because the antibody recognizes the hapten on the targetable construct. In this way, a variety of effectors can be used in the pretargeting system, provided that the targetable construct that includes the effector maintains the same recognized hapten.
Because in a pre-targeting method the effector molecule (i.e., targeting molecule or carrier molecule) and the binding molecule (i.e., the targeting construct or antibody) are not administered concurrently, the binding molecule must not be internalized by the targeted tissue prior to administering the effector molecule. However, because the binding molecule is bivalent and bispecific, internalization of the binding molecule may be hindered or delayed until after the effector molecule is administered, even if the binding molecule recognizes an antigen that is part of an internalizing receptor on the surface of the targeted tissue. Further, if the effector molecule is multivalent (i.e., it has two or more moieties recognized by the binding molecule), the effector molecule can crosslink two or more binding molecules on the surface of the targeted tissue to facilitate internalization of the crosslinked complex. The effector molecule may also include one or more moieties that facilitate internalization by binding to internalizing receptors on the surface of the targeted tissue (e.g., the folate receptor). Methods of compositions for administering therapeutic and diagnostic agents are described in U.S. 60/444,357, filed Jan. 31, 2003.
Thus, there is a continuing need for immunological agents which can be directed to diseased tissue and can specifically bind to a subsequently administered targetable diagnostic or therapeutic conjugate, and a flexible, modular system that accommodates different diagnostic and therapeutic agents without alteration to the bi-specific or multi-specific antibodies. We have continued to develop the pretargeting system originally described by Janevik-Ivanovska et al. that used an antibody directed against a histamine derivative, histamine-succinyl-glycl (HSG), as the recognition system on which a variety of effector substances could be prepared. Excellent pretargeting results have been reported using a radioiodinated and a rhenium-labeled divalent HSG-containing peptide. In the present work, we have expanded this system to include peptides that include haptens and/or chelators such as DTPA, and which may be suitable for radiolabeling with 90Y, 111In, and 177Lu, as well as 99mTc.